Aedes mosquitoes are the vectors of several arboviruses that cause human disease. A better understanding of their reproduction helps to improve their management and contributes insights into the fundamental biology of mosquitoes. During mating, inseminated mosquito females receive seminal fluids and sperm from males that they then store in the spermathecae. In Aedes aegypti and Aedes albopictus, most mated females become resistant to further insemination within 2 h of initial insemination. Although the male seminal fluids are known to be involved in initiating the resistance of inseminated females to further insemination, the mechanism underlying this resistance is not well-understood. The determination of insemination status is a key step in investigating the behavioral and molecular interactions between males and females and for exploring the proximate influences and evolutionary implications of interspecific copulations. Several methods exist for determining insemination status, as discussed here. The choice of method depends on the research question and the availability of resources.